1,720,975 research outputs found
Resistance to methotrexate is associated with selective changes of α2,6- and α2,3-sialyltransferase activities toward N-acetyllactosaminic sequences in human colon cancer cell line HT-29
No full textIn previous works we established that the α2,6-sialyltransferase acting on N-acetyllactosaminic sequences [α2,6(N)ST, E.C. 2.4.99.1] behaves, in colonic cells, as an onco-developmentally regulated enzyme. Subpopulations of the human colon cancer cell line HT-29 adapted to grow in 10-5 M methotrexate (MTX), permanently retain the ability to differentiate as mucus-secreting cells when kept confluent for extended periods of time [Lesuffleur et al. (1991) J. Cell Biol. 115, 1409-1418]. In this study we have compared the activities of five sialyltransferases acting on N- or O-linked chains of glycoproteins in parental HT-29 and in the 10-5 M MTX-resistant variant. Both cell lines were studies during the exponential phase of growth as well as after a long period of postconfluent culture (28-30 days). Regardless the culture conditions, resistance to 10-5 M MTX is associated with a virtual disappearance of α2,6(N)ST activity. This change results in a dramatic reduction of the reactivity of cell membranes with the fluorescent lectin from Sambucus nigra, specific for α2,6-sialylated structures. The activity of the α2,3-dialyltransferase which acts on N-acetyllactosaminic sequences increases about two times in postconfluent cultures of 10-5 M MTX-resistant cells, suggesting a close relationship with the differentiation degree. No significative changes were observed in the activity of other sialyltransferases. © 1993 Academic Press, Inc
Processing of herpes simplex virus-1 glycans in cells defective in glycosyl transferases of the golgi system: Relationship to cell fusion and virion egress
No full textWe studied herpes simplex virus-1 (HSV-1) glycan structure and the expression of HSV-1 functions regulated by viral glycoproteins in Ric21 cells (P. Vischer and R. C. Hughes, Eur. J. Bioch. 117, 275-284, 1981). This is a line of ricin-resistant mutant BHK cells defective in the enzymes of the Golgi system which add terminal sugars to N-linked glycans. Twp kinds of alterations were observed in the glycosylation of HSV glycoproteins in Ric21 cells. First, there was a defective processing of complex glycans leading to a reduction of biantennary and triantennary species and an increase of incompletely processed monosialylated oligosaccharides. Second, there was an overall reduction in the accumulation of HSV-1 glycoproteins. We found that (i) the release of herpesvirions from Ric21 cells was markedly lower than that from BHK cells, possibly reflecting reduced terminal sugar addition which, in turn, might affect the intracellular transport of glycoproteins. (ii) HSV-1 (MP)-infected Ric21 cells fused with a low efficiency. Furthermore, polycaryocytosis was reduced or abolished in BHK and in Ric21 cells exposed to neuraminidase, indicating that the presence of sialic acid residues in the cell surface glycans is essential for cells to interact in a fashion that brings cell fusion. (iii) Although capsid assembly was comparable, the rate of accumulation of infectious virus decreased in Ric21 cells. Infectivity of released virions from Ric21 and BHK cells was similar, in agreement with previous studies showing that complex-type glycans do not appear to be required for herpesvirion infectivity. The decrease in infectious HSV-1 yield seems to correlate with overall reduced ability to synthesize glycoproteins. © 1983
Rapid isolation of Tamm-Horsfall glycoprotein (uromodulin) from human urine
No full textAn isolation method for Tamm-Horsfall protein is described which is based on the observation that a diatomaceous earth filter is able to retain most of the glycoprotein present in urine and that the glycoprotein is easily desorbed from the filter by deionized water. This behaviour depends on the tendency of Tamm-Horsfall glycoprotein at normal urinary concentrations to form a gel in a solution containing mono- and divalent ions. By means of two-step filtration, the glycoprotein was purified to homogeneity. The yield was of about 20 mg/l of urine, and the time required for the isolation was approximately 5-6 h. This procedure should be particularly useful for preparing large amounts of Tamm-Horsfall glycoprotein oligosaccharides in order to investigate their potential use as immunosuppressive agents both in vitro and in vivo. © 1989
Temporal aspects of O-glycosylation of glycoprotein C from herpes simplex virus type-1
No full textHerpes simplex virus type-1 glycoprotein C (gC1) contains several O-linked oligosaccharides clustered near N-linked chains, and Pronase digestion produces glycopeptides carrying both oligosaccharide types. We have taken advantage of this fact to investigate the temporal relationship between the initiation of O-linked chains and the processing of N-linked oligosaccharides. gC1 was isolated from herpes-simplex-virus-infected BHK (baby-hamster kidney) cells after short labelling periods with [3H]glucosamine, and the labelled Pronase-cleaved glycopeptides fractionated on concanavalin A-Sepharose. N-[3H]Acetylgalactosamine, mostly convertible into free N-[3H]acetylgalactosaminitol on mild alkaline-borohydride treatment, was found in glycopeptides with an affinity to concanavalin A-Sepharose corresponding to that of glycopeptides carrying Man8GlcNAc2 or larger N-linked chains. Since there is evidence that the processing of N-linked chains up to Man8GlcNAc2 involves enzymes located in the rough endoplasmic reticulum, current results strongly suggest that gC1 acquires O-linked N-acetylgalactosamine before the glycoprotein routing to the Golgi apparatus. The addition of the second sugar to the nascent O-linked chain appeared to occur after a relatively long lag time
Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC
No full textImmature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-mim pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-β-N-acetylglycosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-β-N-acetylglycosaminidase H-cleaved oligosaccharides, polymannosyl chains of different size, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies
Infectivity and glycoprotein processing of herpes simplex virus type 1 grown in a ricin-resistant cell line deficient in N-acetylglucosaminyl transferase I
No full textWe report on the replication of herpes simplex virus type 1 (HSV-1) and viral glycoprotein processing in Ric(R)14 cells, a mutant ricin-resistant cell line defective in N-acetylglucosaminyl transferase I activity. In these cells HSV-1(MP) and (F) replicated to yields very similar to those in parental BHK cells. The kinetics of HSV-1 adsorption in mutant and in parent cells was also essentially identical. Progeny virions from ricin-resistant and wild-type cells displayed comparable specific infectivities. However, in the mutant cells the efficiency of plating of progeny virus from both Ric(R)14 and BHK cells was reduced. HSV-1(MP) failed to induce syncytia in Ric(R)14 cells either in a plaque assay or after a high-multiplicity infection. Moreover, the fully glycosylated forms of glycoproteins (gB, gC, and gD) were totally absent, and only the partially glycosylated precursors (pgC, pgD, and a triplet in the gB-gA region) accumulated in HSV-1 infected ricin-resistant cells and in herpesvirions made in these cells. Consistent with these results analysis of pronase glycopeptides from cells labeled with [14C]glucosamine showed a strong decrease of sialylated complex-type oligosaccharides and a dramatic accumulation of the neutral mannose-rich chains. The latter chains predominate in partially glycosylated precursors, whereas the complex acidic chains predominate in the fully processed forms of HSV glycoproteins. These results taken together indicate that (i) host-cell N-acetylglucosaminyl transferase I participates in the processing of HSV glycoproteins; and (ii) infectivity of herpesvirions does not necessarily require the mature form of gB. The absence of HSV-1(MP)-induced fusion in Ric(R)14 cells is discussed
Biosynthesis and oligosaccharide structure of human CD8 glycoprotein expressed in a rat epithelial cell line.
No full textPost-translational processing of an O-glycosylated protein, the human CD8 glycoprotein, during the intracellular transport to the plasma membrane.
No full textCharacterization of N- and O-linked oligosaccharides of glycoprotein 350 from Epstein-Barr virus
No full textGlycoprotein 350 (gp350), the major Epstein-Barr Virus (EBV) envelope glycoprotein, has extensive N- and O-linked oligosaccharide chains. To characterize these oligosaccharide chains, [3H]glucosamine-labeled gp350 was isolated from an EBV transformed marmoset lymphoblastoid cell line (B95-8) induced to replicate EBV. Radiolabeled pronase-glycopeptides were fractionated by serial affinity chromatography and O-linked oligosaccharides released by mild alkaline borohydride treatment. Virtually all (99%) N-linked oligosaccharides were of complex type, with a predominance of tri-tetraantennary versus diantennary chains. A significant portion (28%, in term of radioactivity) of the tri-tetraantennary chains bound to leucoagglutinin-agarose, indicating an additional branch in β(1-6)-linkage to the trimannosyl core. N-linked oligosaccharides with such a branching pattern have not been previously described in any herpesvirus glycoprotein, but have been associated with neoplastic transformation. Half of [3H]glucosamine incorporated into gp350 was recovered in O-linked oligosaccharides. The smallest chains have a core βGal-GaINAc disaccharide structure. Most O-linked chains have two to three N-acetylglucosamine and one N-acetylgalactosamine residues, besides the N-acetylgalactosamine residue located at the terminal reducing end, suggesting a di- or tri- N-acetyllactosamine structure. Consistent with such a structure, the size of these chains, after sialic acid removal, was that of an heptasaccharide or larger. © 1989
Biosynthesis and oligosaccharide structure of human CD8 glycoprotein expressed in a rat epithelial cell line
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